1) Field of the Invention
The present invention relates to a method for producing a dipeptide, and particularly relates to a method for producing a peptide simply and inexpensively with a high yield.
2) Description of the Related Art
Peptides have been used in various fields such as pharmaceuticals and foods. For example, L-alanyl-L-glutamine has been widely used as a component of infusion solutions and serum-free media, because it is more stable and more soluble in water than L-glutamine.
As methods for producing the peptides, chemically synthetic methods have been conventionally known, but the methods have not been always simple. Such synthetic methods may include the method using N-benzyloxycarbonylalanine (hereinafter referred to as “Z-alanine”) and protected L-glutamine [Non-patent Document 1: Bull. Chem. Soc. Jpn., 34, 739 (1961); Non-patent Document 2: Bull. Chem. Soc. Jpn., 35, 1966 (1962)], the method using Z-alanine and protected L-glutamic acid-γ-methyl ester [Non-patent Document 3: Bull. Chem. Soc. Jpn., 37, 200)1964)], the method using Z-alanine ester and unprotected glutamic acid (Patent Document 1: JP 1-96194 A), the method of generating an N-(2-substituted)-propionyl glutamate derivative as an intermediate using 2-substituted propionyl halide as a raw material.
However, in any of the methods, introduction/elimination of a protecting group or the use of an optically active intermediate is required. Thus none of these production methods are industrially advantageous and satisfactory.
Meanwhile, typical methods for producing peptides using enzymes may include a condensation reaction using an N-protected and C-unprotected carboxy component and an N-unprotected and C-protected amine component (Reaction 1), and a substitution reaction using an N-protected and C-protected carboxy component and an N-unprotected and C-protected amine component (Reaction 2). Examples of the Reaction 1 may include the method for producing Z-aspartyl-phenylalanine methyl ester from Z-aspartic acid and phenylalanine methyl ester (Patent Document 3: JP 53-92729 A), and examples of the Reaction 2 may include the method for producing acetyl-phenylalanyl-leucineamide from acetyl phenylalanine ethyl ester and leucineamide [Non-patent Document: Biochemical J., 163, 531 (1977)]. There are extremely few reports of the method using the N-unprotected and C-protected carboxy component. An example of a substitution reaction using an N-unprotected and C-protected carboxy component and an N-unprotected and C-protected amine component (Reaction 3) may be found in WO 90/01555 (Patent Document 4), and may include the method for producing arginyl-leucineamide from arginine ethyl ester and leucineamide. Examples of a substitution reaction using an N-unprotected and C-protected carboxy component and an N-unprotected and C-unprotected amine component (Reaction 4) may be found in EP 278787A1 (Patent Document 5) and EP 359399B1 (Patent Document 6), and may include the method for producing tyrosyl alanine from tyrosine ethyl ester and alanine.
The most inexpensive method of production among the above Reaction 1 to Reaction 4 may obviously be reactions categorized in Reaction 4 with the least protecting groups.
However, the prior-art example of the Reaction 4 (in Patent EP 278787A1) has the following crucial problems: (1) Extremely slow peptide-synthesizing rate; (2) Low peptide generation yield; (3) Limitation of the producible peptides to those having relatively high hydrophobicity; (4) Requirement of a large amount of enzymes to be added; and (5) Requirement of a relatively expensive carboxy peptidase preparation derived from fungi, yeasts and plants.
Among the methods falling into the category of the Reaction 4, none of known methods use enzymes derived from bacteria and yeasts other than Saccharomyces, and none of known methods can produce highly hydrophilic peptides such as alanyl-glutamine. In such a situation, development of the industrially inexpensive methods for producing these peptides has been desired.